Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.
Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.
复制标题
通过甘氨酸->丙氨酸突变探测嗜热菌蛋白酶样蛋白酶中的催化铰链弯曲运动。
作者：
O. R. Veltman;V. Eijsink;Gert Vriend;A. D. Kreij;Gerard Venema;B. Burg
热溶酶样蛋白酶（TLPs）的活性位点位于N端和c端结构域之间间隙的底部。晶体学研究表明，与无配体的TLPs相比，结合配体的TLPs的活性位点间隙更封闭。因此，有人提出TLPs在催化过程中经历铰链弯曲运动，导致活性位点间隙的“关闭”和“打开”。提出了两个铰链区域。一个位于保守的甘氨酸78周围；第二个涉及到残数135和136。通过分析Gly -> Ala突变对催化活性的影响，实验研究了这些铰链区域中保守甘氨酸残基的重要性。对嗜热脂肪芽孢杆菌（Bacillus stearotherophilus, TLP-ste）的TLP进行了8次突变，并测定了它们对酪蛋白和各种肽底物活性的影响。只有Gly78Ala、Gly136Ala和Gly135Ala + Gly136Ala突变体的催化活性显著降低。这些突变体显示3-（2-呋喃丙烯酰）- l-甘酰基-l -亮氨酸酰胺的kcat/Km分别降低了73%、62%和96%。对各种底物的kcat/Km的影响与对磷酰胺Ki的影响的比较表明，78位突变主要影响底物结合，而135位和136位突变主要影响kcat。在提出的铰链弯曲区域中，保守的甘氨酸残基对TLP活性的明显重要性支持了铰链弯曲是催化的重要组成部分的观点。
The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.